The macula densa is a unique plaque of cells that are within the cortical thick ascending limb (cTAL) and lie next to the mesangial cell/afferent arteriolar complex. When luminal sodium chloride concentration ([NaCl]L is elevated, macula densa detect this increase and transmit signals that cause vasoconstriction and decreases in blood flow and glomerular filtration rate. This tubuloglomerular feedback (TGF) mechanism is an important regulator of renal hemodynamics. Our work has focused on the nature of this TGF signaling pathway. Recent studies have identified that, an increase in [NaC1]L depolarizes the basolateral membrane potential resulting in calcium entry into macula densa cells through a non-selective cation channel. In addition, an elevation in [NaC1] L also activates a maxichloride (CI) channel at the basolateral membranewhich results in the release of ATP. Since mesangial cells possess purinergic receptors we believe that ATP is a signaling molecule that is involved in communication from macula densa to mesangial cells. To further understand this TGF signaling process, studies will be performed in the isolated perfused cTAL containing the macula densa plaque. Fluorescence microscopy for intracellular ion concentrations, patch-clamp techniques and protein identification using immunofluorescence will be performed. These studies will: 1) further define macula densa-ATP signaling using the newly developed PC12 cell-P2X receptor bioassay. Also studies will be performed to assess the contributions of the various macula densa cell transport pathways to basolateral ATP release, 2) characterize ATP signaling to the underlying mesangial cells and afferent arteriolar smooth muscle cells using state-of-the-art 2 photon confocal microscopy, 3) identify and further characterize the regulation of the recently identified non-selective cation and the maxi Cl channels at the basolateral membrane of macula densa cells, and 4) identify other potential vasoactive agents that may be released by the macula densa cells including nitric oxide, prostglandins, thromboxanes and adenosine. These studies will provide important new insights into this macula densa cell-to-cell TGF signaling pathway which serves a vital role in the regulation of renal hemodynamics.